All but the simplest reactions and assays require multiple steps where new reagents are added between steps. In microtitre-plate based systems, this is achieved by pipetting in new reagents at defined times. However, even using sophisticated (and expensive) robotic liquid-handling systems the throughput is little more than one per second. The quest for higher throughput is, of necessity, driving the development of ever smaller reaction vessels. However, there is little scope to further reduce reaction volumes below the current minimum of 1-2 μl using microtitre plate technology.
One option is to use microdroplets in water-in-oil emulsions as microreactors: the droplets have volumes 103 to 109 times smaller than the smallest working volume in a microtitre plate well. In Vitro Compartmentalization (IVC) (Tawfik and Griffiths, 1998) of reactions in emulsions was initially developed for directed evolution and has allowed the selection of a wide range of proteins and RNAs for binding, catalytic and regulatory activities (Griffiths and Tawfik, 2006). Other applications rapidly followed, notably massively parallel PCR of single DNA molecules (emulsion PCR), which is used, for example, for two commercial ‘next-generation’high-throughput sequencing systems (Mardis, 2008).
However, it is difficult to add reagents to droplets in bulk emulsions after they are formed, which is a serious limitation. This problem can potentially be overcome using droplet-based microfluidic systems, in which controlled pairwise droplet fusion is possible. There are several ways to fuse aqueous droplets within microfluidic channels. Droplets that are not stabilized by surfactant will coalesce spontaneously (Song et al., 2003; Hung et al., 2006; Tan et al., 2007; Niu et al., 2008; Um et al., 2009; Sassa et al., 2008), or can be coalesced based on a surface energy pattern on the walls of a microfluidic device (Fidalgo et al., 2007; Liu and Ismagilov, 2009), or a new stream of fluid can be merged with large droplets passing the orifice (Zheng and Ismagilov, 2005). Surfactant stabilized droplets can be fused using local heating from a focused laser (Baroud et al., 2007) or using electric forces (Link et al., 2006; Priest et al., 2006) and electro-coalescence has been used to measure millisecond enzyme kinetics (Aim et al. 2006) and for the synthesis of magnetic iron oxide nanoparticles (Frenz et al. 2008).
The main problem in droplet coalescence for biological or chemical applications is the existence of two contradictory constraints: first the stability of droplets as microreactors has to be guaranteed and second, these droplets have to be destabilized when necessary. Spontaneous fusion of droplets without surfactant requires careful droplet synchronization and often gives high numbers of undesirable fusion events. In addition, in the absence of surfactant, further droplet manipulations are restricted because the fused droplets are unstable. The use of external force is therefore a straightforward solution but not perfect: coalescence induced by heating with a laser is not suitable for most biological or heat-sensitive chemical reactions and the throughput is limited to ˜10 fusions per second, while electro-coalescence, requires precise droplet synchronization, sophisticated equipment, including micro fluidics chips with integrated electrodes and good electrical shielding to prevent unwanted electro-coalescence. All of these difficulties limit broader droplet-fusion applications, especially if multiple processing steps are necessary.
Hence, to date, droplets have only been fused shortly after formation, and pairwise droplet fusion has only been used to initiate reactions and not to perform multi-step procedures. However, many reactions need to be incubated for certain periods of time, sufficiently long to achieve desirable amounts of product (e.g. to obtain high yields of in vitro translated protein, to amplify DNA, to bind a drug target etc.), before new reagents are added. Therefore what is needed in the art are systems and methods for an efficient and reliable generation system allowing controllable and reliable droplet fusion which can be used to perform multi-step procedures.